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The dystroglycan complex is necessary for stabilization of acetylcholine receptor clusters at neuromuscular junctions and formation of the synaptic basement membrane.

Jacobson C, Côté PD, Rossi SG, Rotundo RL, Carbonetto S - J. Cell Biol. (2001)

Bottom Line: The dystrophin-associated protein (DAP) complex spans the sarcolemmal membrane linking the cytoskeleton to the basement membrane surrounding each myofiber.These results suggest that dystroglycan is essential for the assembly of a synaptic basement membrane, most notably by localizing AChE through its binding to perlecan.In addition, they suggest that dystroglycan functions in the organization and stabilization of AChR clusters, which appear to be mediated through its binding of laminin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University/Center for Neuroscience Research, Montréal General Hospital Research Institute, Montréal, Québec H3G 1A4, Canada.

ABSTRACT
The dystrophin-associated protein (DAP) complex spans the sarcolemmal membrane linking the cytoskeleton to the basement membrane surrounding each myofiber. Defects in the DAP complex have been linked previously to a variety of muscular dystrophies. Other evidence points to a role for the DAP complex in formation of nerve-muscle synapses. We show that myotubes differentiated from dystroglycan-/- embryonic stem cells are responsive to agrin, but produce acetylcholine receptor (AChR) clusters which are two to three times larger in area, about half as dense, and significantly less stable than those on dystroglycan+/+ myotubes. AChRs at neuromuscular junctions are similarly affected in dystroglycan-deficient chimeric mice and there is a coordinate increase in nerve terminal size at these junctions. In culture and in vivo the absence of dystroglycan disrupts the localization to AChR clusters of laminin, perlecan, and acetylcholinesterase (AChE), but not rapsyn or agrin. Treatment of myotubes in culture with laminin induces AChR clusters on dystroglycan+/+, but not -/- myotubes. These results suggest that dystroglycan is essential for the assembly of a synaptic basement membrane, most notably by localizing AChE through its binding to perlecan. In addition, they suggest that dystroglycan functions in the organization and stabilization of AChR clusters, which appear to be mediated through its binding of laminin.

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Dystroglycan- myotubes have AChR clusters on their surface and respond to agrin. Wild-type (R1) and dystroglycan- (3C12 and 3H1) ES cells were differentiated in culture for up to 30 d to generate spontaneously contracting myotubes (A, left). When cells were treated with agrin and stained with TRITC–α-Btx, AChR clusters were observed on the surfaces of all three differentiated ES cell lines. Dystroglycan- myotubes (3C12 and 3H1) have AChR clusters comprised of diffuse arrays of microclusters. This contrasts with the AChR clusters on R1 cells, which are in smaller and more uniformly fluorescent plaques (A, right). (B) Overnight treatments with 500 pM agrin induced an approximately fourfold increase in the number of clusters for all three cell lines compared with untreated cultures. The data presented represent at least three separate experiments with 10–20 microscope fields of myotubes quantified per experiment. Significance was tested using Student's t test. Bar, 10 μm.
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Figure 2: Dystroglycan- myotubes have AChR clusters on their surface and respond to agrin. Wild-type (R1) and dystroglycan- (3C12 and 3H1) ES cells were differentiated in culture for up to 30 d to generate spontaneously contracting myotubes (A, left). When cells were treated with agrin and stained with TRITC–α-Btx, AChR clusters were observed on the surfaces of all three differentiated ES cell lines. Dystroglycan- myotubes (3C12 and 3H1) have AChR clusters comprised of diffuse arrays of microclusters. This contrasts with the AChR clusters on R1 cells, which are in smaller and more uniformly fluorescent plaques (A, right). (B) Overnight treatments with 500 pM agrin induced an approximately fourfold increase in the number of clusters for all three cell lines compared with untreated cultures. The data presented represent at least three separate experiments with 10–20 microscope fields of myotubes quantified per experiment. Significance was tested using Student's t test. Bar, 10 μm.

Mentions: To assess the roles of dystroglycan in the organization of the sarcolemma and the organization of AChRs, ES cells were grown (see Materials and Methods) and allowed to differentiate for up to 30 d. Under these conditions dystroglycan- ES cells differentiated into several cell types, including neurons, blood cells, and myoblasts (Côté, P.D., and S. Carbonetto, unpublished results). By 20 d in culture myoblasts had fused into myotubes with multiple aligned nuclei which expressed myogenin when assayed immunocytochemically (data not shown). Myotubes in cultures of wild-type (R1) and dystroglycan- ES cell lines (3C12 and 3H1) were indistinguishable under phase optics (Fig. 2 A, left) and contracted spontaneously. After incubation with TRITC-labeled α-Btx, wild-type and dystroglycan- myotubes were both found to have AChR clusters, though there was a clear difference in their morphology. AChRs from wild-type (R1) myotubes were in tight, uniformly fluorescent clusters, whereas clusters of AChRs on dystroglycan- myotubes (3C12 and 3H1) were punctate and covered a larger area than those on wild-type myotubes (Fig. 2 A). Similar results were obtained whether clusters had formed spontaneously or were induced by agrin (Fig. 2 A). The small clusters of AChRs appeared similar to the microclusters that first form on the surface of muscle after innervation and which are known to contain dystroglycan (Cohen et al. 1995). Based on these observations, we hypothesized that dystroglycan either was necessary for the condensation of AChR microclusters into larger clusters, or was required for stabilization of preexisting clusters which had formed normally but then dispersed in the absence of dystroglycan.


The dystroglycan complex is necessary for stabilization of acetylcholine receptor clusters at neuromuscular junctions and formation of the synaptic basement membrane.

Jacobson C, Côté PD, Rossi SG, Rotundo RL, Carbonetto S - J. Cell Biol. (2001)

Dystroglycan- myotubes have AChR clusters on their surface and respond to agrin. Wild-type (R1) and dystroglycan- (3C12 and 3H1) ES cells were differentiated in culture for up to 30 d to generate spontaneously contracting myotubes (A, left). When cells were treated with agrin and stained with TRITC–α-Btx, AChR clusters were observed on the surfaces of all three differentiated ES cell lines. Dystroglycan- myotubes (3C12 and 3H1) have AChR clusters comprised of diffuse arrays of microclusters. This contrasts with the AChR clusters on R1 cells, which are in smaller and more uniformly fluorescent plaques (A, right). (B) Overnight treatments with 500 pM agrin induced an approximately fourfold increase in the number of clusters for all three cell lines compared with untreated cultures. The data presented represent at least three separate experiments with 10–20 microscope fields of myotubes quantified per experiment. Significance was tested using Student's t test. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 2: Dystroglycan- myotubes have AChR clusters on their surface and respond to agrin. Wild-type (R1) and dystroglycan- (3C12 and 3H1) ES cells were differentiated in culture for up to 30 d to generate spontaneously contracting myotubes (A, left). When cells were treated with agrin and stained with TRITC–α-Btx, AChR clusters were observed on the surfaces of all three differentiated ES cell lines. Dystroglycan- myotubes (3C12 and 3H1) have AChR clusters comprised of diffuse arrays of microclusters. This contrasts with the AChR clusters on R1 cells, which are in smaller and more uniformly fluorescent plaques (A, right). (B) Overnight treatments with 500 pM agrin induced an approximately fourfold increase in the number of clusters for all three cell lines compared with untreated cultures. The data presented represent at least three separate experiments with 10–20 microscope fields of myotubes quantified per experiment. Significance was tested using Student's t test. Bar, 10 μm.
Mentions: To assess the roles of dystroglycan in the organization of the sarcolemma and the organization of AChRs, ES cells were grown (see Materials and Methods) and allowed to differentiate for up to 30 d. Under these conditions dystroglycan- ES cells differentiated into several cell types, including neurons, blood cells, and myoblasts (Côté, P.D., and S. Carbonetto, unpublished results). By 20 d in culture myoblasts had fused into myotubes with multiple aligned nuclei which expressed myogenin when assayed immunocytochemically (data not shown). Myotubes in cultures of wild-type (R1) and dystroglycan- ES cell lines (3C12 and 3H1) were indistinguishable under phase optics (Fig. 2 A, left) and contracted spontaneously. After incubation with TRITC-labeled α-Btx, wild-type and dystroglycan- myotubes were both found to have AChR clusters, though there was a clear difference in their morphology. AChRs from wild-type (R1) myotubes were in tight, uniformly fluorescent clusters, whereas clusters of AChRs on dystroglycan- myotubes (3C12 and 3H1) were punctate and covered a larger area than those on wild-type myotubes (Fig. 2 A). Similar results were obtained whether clusters had formed spontaneously or were induced by agrin (Fig. 2 A). The small clusters of AChRs appeared similar to the microclusters that first form on the surface of muscle after innervation and which are known to contain dystroglycan (Cohen et al. 1995). Based on these observations, we hypothesized that dystroglycan either was necessary for the condensation of AChR microclusters into larger clusters, or was required for stabilization of preexisting clusters which had formed normally but then dispersed in the absence of dystroglycan.

Bottom Line: The dystrophin-associated protein (DAP) complex spans the sarcolemmal membrane linking the cytoskeleton to the basement membrane surrounding each myofiber.These results suggest that dystroglycan is essential for the assembly of a synaptic basement membrane, most notably by localizing AChE through its binding to perlecan.In addition, they suggest that dystroglycan functions in the organization and stabilization of AChR clusters, which appear to be mediated through its binding of laminin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University/Center for Neuroscience Research, Montréal General Hospital Research Institute, Montréal, Québec H3G 1A4, Canada.

ABSTRACT
The dystrophin-associated protein (DAP) complex spans the sarcolemmal membrane linking the cytoskeleton to the basement membrane surrounding each myofiber. Defects in the DAP complex have been linked previously to a variety of muscular dystrophies. Other evidence points to a role for the DAP complex in formation of nerve-muscle synapses. We show that myotubes differentiated from dystroglycan-/- embryonic stem cells are responsive to agrin, but produce acetylcholine receptor (AChR) clusters which are two to three times larger in area, about half as dense, and significantly less stable than those on dystroglycan+/+ myotubes. AChRs at neuromuscular junctions are similarly affected in dystroglycan-deficient chimeric mice and there is a coordinate increase in nerve terminal size at these junctions. In culture and in vivo the absence of dystroglycan disrupts the localization to AChR clusters of laminin, perlecan, and acetylcholinesterase (AChE), but not rapsyn or agrin. Treatment of myotubes in culture with laminin induces AChR clusters on dystroglycan+/+, but not -/- myotubes. These results suggest that dystroglycan is essential for the assembly of a synaptic basement membrane, most notably by localizing AChE through its binding to perlecan. In addition, they suggest that dystroglycan functions in the organization and stabilization of AChR clusters, which appear to be mediated through its binding of laminin.

Show MeSH
Related in: MedlinePlus